The long-term objective of this project will be to determine the biochemical and physiological mechanisms of corneal scar formation with regard to the two major macromolecular constituents of the cornea (i.e., collagen and proteoglycans). This will enable us to determine which chemical properties of collagen and proteoglycans are significant in determining the mechanical strength and opacity of scarred corneal tissue, and what regulatory factors control the deposition of these macromolecules. Ultimately, we hope this knowledge can lead to methods for altering the healing process in humans so that transparency can be restored to injured corneas. The specific aim of this research is to compare the morphogenetic processes in healing and fetal corneas with respect to collagen, proteoglycans and fibronectin. Morphological studies: a. Localization and interactions of the macromolecules within healing and fetal corneas will be determined by ultrastructural immunocytochemical, cytochemical and histochemical methods. Monoclonal antibodies and colloidal gold coated with secondary antibodies or protein A will allow localization of macromolecules within the dense corneal stroma. Cytochemical and histochemical methods will complement the immunocytochemical methods. b. Identification of the cell source of collagens, proteoglycans and fibronectin will necessitate development of complementary DNA probes, radiolabeled or conjugated to a biotin-avidin system to hybridize in situ with specific mRNAs from corneal cells. Biochemical studies: These studies will further characterize corneal macromolecules and their interactions in vitro. a. Monoclonal antibodies will be used to do peptide mapping analyses with immunoblotting techniques. b. These antibodies, in conjunction with affinity chromatographic techniques, will also be used to study the interactions of corneal macromolecules. c. In addition, the antibodies will help isolate and purify in vitro-translating mRNA species. d. cDNAs will aid in sequencing amino acids of corneal proteoglycans, collagen and fibronectin and act as probes to localize, isolate and quantify specific mRNAs from different stages of fetal and healing corneal tissues. e. Corneal organ culture techniques will be developed to study regulation of corneal macromolecular synthesis. These studies are primary to the development of methods for modulating corneal wound healing.